This application is a national stage filing of PCT International Application No. PCT/FI01/00241, filed on Mar. 12, 2001. This application also claims the benefit of priority under 35 U.S.C. xc2xa7 119(a) to Finnish patent application No. 20000577, filed on Mar. 13, 2000.
The present invention relates to pyridazinyl phenyl hydrazone compounds and pharmaceutically acceptable salts and esters thereof. The invention also relates to pharmaceutical compositions comprising such compounds as active ingredients. The compounds of the invention increase the calcium sensitivity of contractile proteins of the cardiac muscle and are thus useful in the treatment of congestive heart failure.
Congestive heart failure is characterized by a decrease in cardiac output and an increase in right and left ventricular filling pressure. These hemodynamic conditions can produce symptoms of dyspnea, fatigue and edema.
The contraction in cardiac muscle is triggered by the binding of calcium to contractile proteins. Series of phosphodiesterase isoenzyme III (PDE III) inhibitors are in clinical trials for the treatment of congestive heart failure. These compounds increase the contractility of the cardiac muscle and produce vasodilatation. However, it is known that the long-term application of those compounds may lead to calcium overload in the cardiac muscle and trigger arrhythmias. It is therefore desired to develop medicaments acting by a mechanism which would increase cardiac contractility without producing calcium overload. The increase of calcium sensitivity of contractile proteins would be such a mechanism.
Pyridazinyl phenyl hydrazone compounds have been described earlier in European patent application EP 383449. The compounds show calcium dependent binding to contractile proteins of the cardiac muscle, as well as PDE III inhibiting activity. In the specific examples one 1-acetyl-1-phenyl methylidene derivative is disclosed (Ex. 16). While the 1-acetyl-1-phenyl methylidene derivative has some effect in cardiac contractility, it does not increase the calcium sensitivity of contractile proteins.
Certain pyridazinyl phenyl hydrazone compounds appear as intermediates in European patent applications EP 223937 and EP 280224. However, the compounds are not specifically characterized. Mertens, A. et al., J. Med. Chem. 1990, 33, 2870-2875,discloses a phenyl, 4-methoxyphenyl and 2-hydroxyphenyl derivatives of pyridazinyl phenyl hydrazone compounds as intermediates.
It has now been found that compounds of formula (I) are potent in increasing the calcium sensitivity of contractile proteins in the cardiac muscle: 
in which
R1 to R4 means hydrogen, alkyl, alkenyl, aryl, arylalkyl, carboxyalkyl, hydroxyalkyl or halogenalkyl, or R2 and R3 form a ring of 5-7 carbon atoms,
R5 to R9 means hydrogen, alkyl, alkenyl, aryl, arylalkyl, acyl, hydroxy, alkoxy, alkoxycarbonyl, amino, acylamino, alkylamino, aryloxy, halogen, cyano, nitro, carboxy, alkylsulfonyl, sulfonamido or trifluoromethyl,
wherein each aryl residue defined above by itself or as a part of another group may be substituted,
and pharmaceutically acceptable salts and esters thereof,
provided that a) when R1, R2, R3, R5, R6, R8 and R9 are hydrogen and R4 is methyl, R7 is not hydrogen or methoxy and b) when R1, R2, R3, R5, R6, R7 and R8 are hydrogen and R4 is methyl, R9 is not hydroxy.
The invention also relates to compounds of formula (I) in which R1, R2, R3, R5, R6, R8 and R9 are hydrogen, R4 is methyl, and R7 is hydrogen or methoxy, or in which R1, R2, R3, R5, R6, R7 and R8 are hydrogen, R4 is methyl and R9 is hydroxy and pharmaceutically acceptable salts and esters thereof, for use as a medicament.
In a class of preferred compounds and pharmaceutically acceptable salts and esters are compounds of formula (I) wherein R5 to R9 are independently hydrogen, C1-6alkyl, C1-6alkenyl, C6-10aryl, C7-12arylalkyl, C1-6acyl, hydroxy, C1-6alkoxy, C1-6alkoxycarbonyl, amino, C1-6acylamino, C1-6alkylamino, C6-10aryloxy, halogen, cyano, nitro, carboxy, C1-6alkylsulfonyl, sulfonamido or trifluoromethyl. In a subclass of this class of compounds and pharmaceutically acceptable salts thereof are compounds of formula (I) wherein R5 to R9 are independently hydrogen, hydroxy, C1-6alkyl, C1-6alkoxy, carboxy, C1-6alkoxycarbonyl or nitro. In a subclass of this class of compounds and pharmaceutically acceptable salts thereof are compounds of formula (I) wherein R5 is hydroxy, C1-6alkyl, C1-6alkoxy, carboxy, C1-6alkoxycarbonyl or nitro, most preferably hydroxy or nitro.
In another class of preferred compounds and pharmaceutically acceptable salts R1 to R4 are independently hydrogen, C1-6alkyl, C1-6alkenyl, C6-10aryl, C7-12arylalkyl, C1-6carboxyalkyl, C1-6hydroxyalkyl or C1-6halogenalkyl, or R2 and R3 form a phenyl ring. In a subclass of this class of compounds and pharmaceutically acceptable salts thereof are compounds of formula (I) wherein R1 to R3 are independently hydrogen or C1-6alkyl.
Each aryl residue in each of these preferred classes of compounds, by itself or as part of another group, may be substituted by 1 to 3, preferably 1 or 2, of fluorine, chlorine, bromine, iodine, hydroxy, nitro, carboxy, trifluoromethyl, amino, C1-4alkyl, C1-4alkoxy, C1-6acyl, C1-6carboxyalkyl, phenyl, naphthyl, halophenyl, halonaphthyl, benzyl, phenethyl, halobenzyl, halophenethyl, naphthylmethyl, naphthylethyl, C4-7cycloalkyl, C1-4alkyl C4-7cycloalkyl, mono C1-4alkylamino, di C1-4alkylamino, C1-6alkanoylamino, phenylcarbonylamino, naphthylcarbonylamino, cyano, thiol, or C1-6alkylthio.
The compounds of formula (I) may contain one or more assymmetric centers and thus they can exist as enantiomers or diastereomers. The invention includes both mixtures and separate individual isomers.
Especially preferred individual compounds of the invention include:
(R)-6-{4-[Nxe2x80x2-(4-Hydroxy-3-methoxy-2-nitro-benzylidene)-hydrazino]-phenyl }-5-methyl-4,5-dihydro-2H-pyridazin-3-one;
6-{4-[Nxe2x80x2-(4-Hydroxy-3-methoxy-2-nitro-benzylidene)-hydrazino]-phenyl}-5-methyl-4,5-dihydro-2H-pyridazin-3-one;
6-(4-{Nxe2x80x2-[1-(2,5-Dihydroxy-phenyl)-ethylidene]-hydrazino}-phenyl)-5-methyl-4,5-dihydro-2H-pyridazin-3-one;
6-(4-{N-[1-(2,4-Dihydroxy-3-methylphenyl)ethylidene]hydrazino}phenyl)-5-methyl-4,5-dihydro-2H-pyridazin-3-one;
6-(4-{Nxe2x80x2-[Bis-(2,4-dihydroxy-phenyl)-methylene]-hydrazino}-phenyl)-5-methyl-4,5-dihydro-2H-pyridazin-3-one;
6-(4-{Nxe2x80x2-[1-(2,4-Dihydroxy-phenyl)-ethylidene]-hydrazino}-phenyl)-5-methyl-4,5-dihydro-2H-pyridazin-3-one;
2,6-Dihydroxy-3-{[4-(4-methyl-6-oxo-1,4,5,6-tetrahydro-pyridazin-3-yl)-phenyl]-hydrazonomethyl }-benzoic acid ethyl ester; and
6-{4-[Nxe2x80x2-(3-Ethyl-2,4-dihydroxy-benzylidene)-hydrazino]-phenyl}-5-methyl-4,5-dihydro-2H-pyridazin-3-one.
The compounds of the invention can be prepared by the well known condensation reaction between a carbonyl compound and a hydrazine as shown in Scheme 1: 
wherein Ar means 
and R1 to R9 as defined above.
A suitable method for the preparation of hydrazines (III) is the diazotization of an aniline and reduction as a one pot synthesis. Scheme 2 shows this reaction: 
where Ar is as above.
Compounds of formula (II) and (IV) are commercially available or can be prepared using methods known in the literature.
General method 1: In case where R4 is hydrogen, the reaction of Scheme 1 is generally performed by refluxing a mixture of compounds (II) and (III) in a suitable solvent, such as ethanol, 2-propanol, acetonitrile or acetic acid, for 1-24 hours. The product (I) is filtered.
General method 2: In case where R4 is not hydrogen, the reaction of Scheme 1 is generally performed by heating a neat mixture of compounds (II) and (III) at 140-170xc2x0 C. under inert atmosphere. The mixture is then triturated with ethyl acetate and the product (I) filtered.
Salts and esters of the compounds, when applicable, may be prepared by known methods. Physiologically acceptable salts are useful as active medicaments, however, preferred are the salts with alkali or alkaline earth metals. Physiologically acceptable esters are also useful as active medicaments. Examples are the esters with aliphatic or aromatic alcohols.
The term xe2x80x9calkylxe2x80x9d as employed herein by itself or as part of another group includes both straight, branched and cyclized chain radicals of up to 18 carbon atoms, preferably 1 to 8 carbon atoms, most preferably 1 to 4 carbon atoms. The term xe2x80x9clower alkylxe2x80x9d as employed herein by itself or as part of another group includes straight, branched and cyclized chain radicals of 1 to 7,preferably 1 to 4,most preferably 1 or 2 carbon atoms. Specific examples for the alkyl and lower alkyl residues, respectively, are methyl, ethyl, propyl, isopropyl, butyl, tert. butyl, pentyl, cyclopentyl, hexyl, cyclohexyl, octyl, decyl and dodecyl including the various branched chain isomers thereof.
The term xe2x80x9cacylxe2x80x9d as employed herein by itself or as part of another group refers to an alkylcarbonyl or alkenylcarbonyl group, the alkyl and alkenyl groups being defined above.
The term xe2x80x9carylxe2x80x9d as used herein by itself or as part of another group refers to a monocyclic or bicyclic group containing from 6 to 10 carbon atoms in the ring portion. Specific examples for aryl groups are phenyl, naphtyl and the like. xe2x80x9cAroylxe2x80x9d means in a corresponding way an arylcarbonyl group.
The term xe2x80x9calkoxyxe2x80x9d as employed herein by itself or as part of another group includes an alkyl group as defined above linked to an oxygen atom. xe2x80x9cAryloxyxe2x80x9d means in a corresponding way an aryl group linked to an oxygen atom.
The term xe2x80x9csubstitutedxe2x80x9d as used herein in connection with various residues refers to halogen substituents, such as fluorine, chlorine, bromine, iodine or trifluoromethyl group, amino, alkyl, alkoxy, aryl, alkyl-aryl, halogen-aryl, cycloalkyl, alkylcycloalkyl, hydroxy, alkylamino, alkanoylamino, arylcarbonylamino, nitro, cyano, thiol, or alkylthio substituents.
The xe2x80x9csubstitutedxe2x80x9d groups may contain 1 to 3,preferably 1 or 2 of the above mentioned substituents.
Compounds of the invention may be administered to a patient in therapeutically effective amounts which range usually from about 0.1 to 500 mg per day depending on the age, weight, condition of the patient, administration route and the phospholamban inhibitor used. The compounds of the invention can be formulated into dosage forms using the principles known in the art. It can be given to a patient as such or in combination with suitable pharmaceutical excipients in the form of tablets, dragees, capsules, suppositories, emulsions, suspensions or solutions. Choosing suitable ingredients for the composition is a routine for those of ordinary skill in the art. It is evident that suitable carriers, solvents, gel forming ingredients, dispersion forming ingredients, antioxidants, colours, sweeteners, wetting compounds and other ingredients normally used in this field of technology may be also used. The compositions containing the active compound can be given enterally or parenterally, the oral route being the preferred way. The contents of the active compound in the composition is from about 0.5 to 100%, preferably from about 0.5 to about 20%, per weight of the total composition.
The usefulness of the compounds of the invention is demonstrated by the following experiments.
Experiment 1. Calcium sensitizing effect in skinned cardiac fiber
Method
The heart of a guinea-pig was excised and perfused with ice-cold saponin (125 mg/l) skinning solution consisting of (mM): K+-acetate 74.7. EGTA-Na2 10, MgSO4 5.4, ATP-Na2 4, MOPS 20, pH 7.0 (by 1 M KOH). Left ventricular papillary muscle was dissected and sonicated at 10 Watt for 60 s. The distance between ultrasound probe and the papillary muscle was 10 mm. The fibres ( less than 200 xcexcm in diameter) were dissected from the surface of sonicated papillary muscles in the same solution.
The fibre was glued between platinum wires, one attached to an isometric force transducer (type AE-801,SensoNor, Horten, Norway) and another to a micro manipulator. The fibre was relaxed in a solution consisting of (mM): EGTA-Na2 10, MgSO4 5.4, ATP-Na2 4, MOPS 20. The pH of the solution was adjusted to 7.0 and ionic strength to 0.16 M by the addition of KOH and K+-acetate. Creatine kinase and creatine phosphate were not added as an ATP generating system because the developed tension was well sustained for the time required for experiment. The calculations for ionic strength and for free calcium (pCa 7.0-6.2) were performed using a suitable program. The fibres were stretched in relaxing solution until resting tension was just noticeable. When the calcium (pCa 6.0 or 6.2)-induced tension had reached steady state the test compound (final concentrations 0.1, 0.3, 1, 3,and 10 xcexcM) was cumulatively added into the solution at 6 min intervals. All the experiments were carried out with fresh fibres at normal room temperature.
Results
The calcium sensitizing effect of the compounds are shown in Table 1.
Experiment 2. Effect in left ventricular pressure derivatives in isolated heart
After sacrification the heart of a guinea-pig was rapidly excised and rinsed in oxygenated perfusion buffer. A cannula was inserted into the aorta and secured with a ligature. Retrograde perfusion began as soon as the heart was placed in a thermostatically controlled moist chamber of the Langendorff apparatus (Hugo Sachs Elektronik, KG). Modified Tyrode solution (37xc2x0 C.), equilibrated in a thermostatically controlled bulb oxygenator with carbogen (95% O2 and 5% CO2), was used as a perfusion buffer. The composition of the Tyrode solution was (in mM): NaCl 135; MgCl2xc3x976H2O 1; KCl 5; CaCl2xc3x972H2O 2; NaHCO3 15; Na2HPO4xc3x972H2O 1; glucose 10; pH 7.3-7.4. The perfusion buffer was delivered at the top of the oxygenator by a pump and driven automatically by its controller. Subsequently, the buffer was delivered into the bulbs of the oxygenator chamber by a rotating disk. It was dispersed by making a thin fluid film on a large inner oxygenator surface in O2/CO2 atmosphere leading to saturation of the perfusate with oxygen (partial pressure 660 mmHg at 37xc2x0 C.).
The experiments were carried out under constant pressure condition (50 mmHg). After a short prestabilization (10 min) a latex balloon (size 4) was carefully placed into the left ventricle through the left pulmonary vein and the left atrium. The latex balloon was attached to a stainless-steel cannula coupled with a pressure transducer. The latex balloon, the cannula and the chamber of the pressure transducer were carefully filled with ethylene glycol/water (1:1) mixture avoiding any air-bubble. The isovolumetric left ventricular pressure was recorded through the pressure transducer. At the beginning of the experiment, the volume of the balloon was adjusted to obtain a diastolic pressure of approximately 5 mmHg. Before starting the experiment the heart was allowed to stabilise further for 30-50 min. The systolic and end-diastolic left ventricular pressures were recorded for calculating the maximal positive and negative derivatives of the left ventricular pressure.
Results
The EC50 values (xcexcM) of various compounds of the invention on maximal positive derivative of the left ventricular systolic pressure are shown in Table 2.
To further illustrate the invention, but not by way of limitation, the following examples are provided. The melting points were determined on a Reichert plate melting point apparatus and were not corrected. NMR-spectra were recorded on using a Bruker ARX 400 spectrometer with internal TMS as the reference (0 ppm).